title: Identification of potential sites for tryptophan oxidation in recombinant antibodies using tert-butylhydroperoxide and quantitative LC-MS.
abstract: Amino acid oxidation is known to affect the structure, activity, and rate of degradation of proteins. Methionine oxidation is one of the several chemical degradation pathways for recombinant antibodies. In this study, we have identified for the first time a solvent accessible tryptophan residue (Trp-32) in the complementary-determining region (CDR) of a recombinant IgG1 antibody susceptible to oxidation under real-time storage and elevated temperature conditions. The degree of light chain Trp-32 oxidation was found to be higher than the oxidation level of the conserved heavy chain Met-429 and the heavy chain Met-107 of the recombinant IgG1 antibody HER2, which have already been identified as being solvent accessible and sensitive to chemical oxidation. In order to reduce the time for simultaneous identification and functional evaluation of potential methionine and tryptophan oxidation sites, a test system employing tert-butylhydroperoxide (TBHP) and quantitative LC-MS was developed. The optimized oxidizing conditions allowed us to specifically oxidize the solvent accessible methionine and tryptophan residues that displayed significant oxidation in the real-time stability and elevated temperature study. The achieved degree of tryptophan oxidation was adequate to identify the functional consequence of the tryptophan oxidation by binding studies. In summary, the here presented approach of employing TBHP as oxidizing reagent combined with quantitative LC-MS and binding studies greatly facilitates the efficient identification and functional evaluation of methionine and tryptophan oxidation sites in the CDR of recombinant antibodies.
authors.affiliation: Pharma Development Penzberg, Roche Diagnostics GmbH, Penzberg, Germany.
authors.forename: Miriam
authors.initials: M
authors.lastname: Hensel
authors.forename: Rebecca
authors.initials: R
authors.lastname: Steurer
authors.forename: Juergen
authors.initials: J
authors.lastname: Fichtl
authors.forename: Carsten
authors.initials: C
authors.lastname: Elger
authors.forename: Frank
authors.initials: F
authors.lastname: Wedekind
authors.forename: Andreas
authors.initials: A
authors.lastname: Petzold
authors.forename: Tilman
authors.initials: T
authors.lastname: Schlothauer
authors.forename: Michael
authors.initials: M
authors.lastname: Molhoj
authors.forename: Dietmar
authors.initials: D
authors.lastname: Reusch
authors.forename: Patrick
authors.initials: P
authors.lastname: Bulau
mesh_terms: D000595:Amino Acid Sequence; D000906:Antibodies / Q000737:chemistry / Q000378:metabolism*; D002853:Chromatography, Liquid; D013058:Mass Spectrometry; D008715:Methionine / Q000378:metabolism; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction / Q000187:drug effects; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism*; D012015:Reference Standards; D020349:Surface Plasmon Resonance; D014364:Tryptophan / Q000378:metabolism*; D014466:Ultraviolet Rays; D020122:tert-Butylhydroperoxide / Q000494:pharmacology*
chemical_list: D000906:Antibodies; D011994:Recombinant Proteins; D014364:Tryptophan; D020122:tert-Butylhydroperoxide; D008715:Methionine